The endogenous peptide endothelin-1 (ET-1) is essential for pain and the sensitization of pain fibers to non-noxious stimuli after nerve injury, incision, inflammation and in cancer. In this proposal we seek to understand the relationship between the elevation of ET-1 in skin after injury and inflammation and the ensuing pain. Recent work suggests that ET-1 causes elevated pain in the periphery by acting on tissues that surround nerve endings, rather than directly on nerves; ET-1 and its cognate, GPC receptors ETA and ETB are elevated in conditions of hyperalgesia and allodynia. Antagonists of the ET receptors are able to prevent or reverse these pains, testifying to an important role for endogenous ET-1 in driving chronic pain states. Current evidence indicates that ET-1's actions during cutaneous injury/inflammation is closely linked to three other molecules/ receptors; TRPV1 (the receptor for hot peppers that is also activated by heat and by a H+), glutamate, and CGRP. TRPV1's participation in cutaneous pain from ET-1 or CFA injection into the rat's paw is evident within a very short time and immunocytochemical staining of TRPV1 in cutaneous nerve fibers increases 5-10-fold. In ~30 min after ET-1 or CFA injection, glutamate and CGRP contribute to ET-1's pain sensitizing effects; these substances are also released by cultured sensory neurons in a way that is potentiated by ET-1. This proposal contains 4 Specific Aims to address the processes underlying these changes: 1. To determine the mechanism for the rapid increase in TRPV1 in epidermal nociceptors following injection of ET-1 and local inflammation. 2. To investigate the temporal relationship between the rapid changes in ETA receptor distribution in skin (after inflammation and ET- 1 delivery) and the resulting desensitization of responses to local ET-1. 3. To determine if the ET receptors in skin cells are able to modulate keratinocye TRPV1, and other TRP channels (TRPV3, TRPV4). 4. To evaluate the role of ET-1-induced release of cutaneous glutamate, CGRP and ATP in ET-1- induced algesia and allodynia. Experiments will integrate the results from behavioral, immunocytochemical, biochemical and cell physiological techniques, for determining the importance of different pathways and substances for ET-1-induced pain, for quantitating ET-1-induced changes in TRPV receptors and ET receptors in skin, for establishing the ability of ET-Rs in keratinocytes to modulate TRPV receptors in those cells, and for examining ET-1's ability to stimulate release of pain-inducing substances from the skin. PUBLIC HEALTH RELEVANCE: The results of this research will lead to a better understanding of the role of endothelin and its receptors in elevated pain from nerve injury, from incision (trauma) and from cancer, situations where it is known to contribute substantially. This understanding will provide a basis for designing new drugs and new interventional procedures to reduce or abolish such pain, and so to lessen suffering and improve the quality of life for millions.